Determination Of Hydroxyproline Research Articles

1H-NMR-based metabolomics to dissect the traditional Chinese medicine promotes mesenchymal stem cell homing as intervention in liver fibrosis in mouse model of Wilson's disease.

We administered Bushen Huoxue Huazhuo Formula (BSHXHZF) and transplanted bone marrow mesenchymal stem cells (BMSCs) into mice with Wilson's disease (WD)-related liver fibrosis to evaluate the liver-protecting mechanism of this prescription. Mice, randomly divided into different treatment groups, showed histopathological changes and degree of hepatocyte apoptosis. For hepatic hydroxyproline (Hyp) determination, transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7) mRNA and protein were measured. Chemical profiling of the extract of BSHXHZF using The liquid chromatography-mass spectrometry (LC-MS/MS) and revealing its antifibrosis mechanism using metabolomics. TCM+BMSC group livers exhibited few inflammatory cells. TUNEL revealed abundant brown apoptotic cells in model control groups, while the TCM+BMSC groups showed a significant increase in blue negative expression of liver cells. Hyp in toxic milk (TX) mice groups was significantly lower than that in model control groups (MG). Compared with MG, TGF-β1 expression was significantly lower than all other groups, while BMP-7 expression was significantly higher. Metabolic analysis identified 20 potential biomarkers and 10 key pathways, indicating that BSHXHZF+BMSC intervention has a significant regulatory effect on metabolic disorders of these small molecule substances. BSHXHZF combined with BMSCs can inhibit liver fibrosis and hepatocyte apoptosis by improving related metabolic disorders, and achieving therapeutic effects in WD-related liver fibrosis.

Response of Carrot (Daucus carota L.) to Multi-Contaminated Soil from Historic Mining and Smelting Activities.

A pot experiment was undertaken to investigate the effect of Cd, Pb and Zn multi-contamination on the physiological and metabolic response of carrot (Daucus carota L.) after 98 days of growth under greenhouse conditions. Multi-contamination had a higher negative influence on leaves (the highest Cd and Zn accumulation) compared to the roots, which showed no visible change in terms of anatomy and morphology. The results showed the following: (i) significantly higher accumulation of Cd, Zn, and Pb in the multi-contaminated variant (Multi) compared to the control; (ii) significant metabolic responses-an increase in the malondialdehyde content of the Multi variant compared to the control in the roots (by 20%), as well as in the leaves (by 53%); carotenoid content in roots decreased by 31% in the Multi variant compared with the control; and changes in free amino acids, especially those related to plant stress responses. The determination of hydroxyproline and sarcosine may reflect the higher sensitivity of carrot leaves to multi-contamination in comparison to roots. A similar trend was observed for the content of free methionine (significant increase of 31% only in leaves); (iii) physiological responses (significant decreases in biomass, changes in gas-exchange parameters and chlorophyll a); and (iv) significant changes in enzymatic activities (chitinase, alanine aminopeptidase, acid phosphatase) in the root zone.

Assessment of collagen content in fish skin - development of a flow analysis method for hydroxyproline determination.

This work describes the development of a flow injection method to determine hydroxyproline (HYP), one of collagen's most abundant amino acids. Collagen is a protein with several applications and high nutritional value. Evaluating the feasibility of using collagen from fish skin over its mammalian source is essential. The determination of HYP requires the pre-treatment and hydrolysis of the fish skin to break down collagen into its amino acids, and the HYP value quantified relates to the collagen content. The determination was based on the HYP oxidation with permanganate in an alkaline medium and the consequent decrease of colour intensity registered. Under optimal conditions, the developed method enables the determination of the HYP within the dynamic range of 23.8 to 500 mg L-1, with a limit of detection (LOD) of 2.6 mg L-1 and a limit of quantification (LOQ) of 23.8 mg L-1. Different samples were processed, and the digests were analysed by the proposed method and with the conventional procedure with good correlation (relative error < 7%). Moreover, the analyte quantification is performed faster, simpler, and more accurately, with less toxic solutions. The reproducibility of the developed method was also evaluated by calculating the relative standard deviation of the calibration curve slope (RSD < 1%).

Age-related changes in the ratio of Type I/III collagen and fibril diameter in mouse skin.

The content of type I collagen (COL-I) and type III collagen (COL-III) and the ratio between them not only affect the skin elasticity and mechanical strength, but also determine the fibril diameter. In this research, we investigated the age-related changes in COL-I/COL-III ratio with their formed fibril diameter. The experimental result was obtained from high performance liquid chromatography-mass spectrometer, hydroxyproline determination, picrosirius red staining and transmission electron microscopes (TEM), respectively. The result indicated that the COL-I/COL-III ratio in mouse skin increased with aging. From the 0th to 9th week, the COL-I/COLIII ratio increased from 1.3:1 to 4.5:1. From the 9th to the 18th week, it remained between 4.5:1 and 4.9:1. The total content of COL-I and COL-III firstly increased and then decreased with aging. The TEM result showed that the fibril diameter increased with aging. From the 0th to 9th week, the average fibril diameter increased from 40 to 112 nm; From the 9th to 18th weeks, it increased from 112 to 140 nm. After the 9th week. The fibril diameter showed obvious uneven distribution. Thus, the COL-I/COLIII ratio was proportional to the fibril diameter, but inversely proportional to the uniformity of fibril diameter.

English

Adansonia digitata is a tree plant used in west African local pharmacopoeia. This work aimed to evaluate the wound healing properties of its trunk bark&rsquo;s hydroethanolic extract in wistar rats. The model of excision wound was used. Wistar rats were treated post-excision topically with neutral carbopol gel (Control), carbopol gels containing 2.5 and 5% of A. digitata hydroethanolic trunk bark extract (ADHE) and L-Mesitran. Pictures were taken post excision for analysis. Biopsies were realized for histological examination of the skin excised and for hydroxyproline determination. In vitro membrane stabilization anti-inflammatory test, DPPH and FRAP antioxidant tests, and phytochemical tests were performed. The results show a better rate of contraction in ADHE 2.5 and 5% animals (+56 &plusmn; 0.52% and +68 &plusmn; 0.24% respectively) as earlier as on day three post-excision. The hydroxyproline assay confirmed a better collagen production in ADHE 2.5% and 5% animals (+102.5 &plusmn; 2.9%, and +107 &plusmn; 3.0% respectively). Histological analysis showed a rapid re-epithelialization in ADHE animals. The anti-inflammatory test indicated an IC50 of 16.521 &plusmn; 0.525 &micro;g/mL, very close to Aspirin (16.826 &plusmn; 0.341 &micro;g/mL), showing a possible anti-inflammatory activity of ADHE. The extract reduced DPPH radical (IC50 of 344.985 &plusmn; 17.139 &micro;g/mL) and ferric ions (EC50 = 178.689 &plusmn; 0.250 &micro;g/mL). Phytochemical investigations revealed the presence of alkaloids, terpenoids, and polyphenolic compounds. Flavonoids in particular, with an amount of 51.694 &plusmn; 0.174 mgER/g, should explain the wound healing activity observed, making the hydroethanolic extract of A. digitata a potential remedy for excision wounds. Key words: Adansonia digitata, wound healing, Wistar rat, antioxidant, anti-inflammatory.

Phagocytosis of alveolar macrophages in experimental animals exposed to chrosotil– asbestos dust

Introduction. The exposure to dust, including chrysotile asbestos, is known to lead to the mobilization of alveolar macrophages, accompanied by the activation of free radical oxidation and the release of mediators stimulating fibroblast proliferation and collagen synthesis. Material and methods. Thirty outbred male rats were divided into two groups: 1 - control with a period of 4 months (n = 15), the 2-experienced group subjected to 4-month seed with chrysotile asbestos dust (n = 15). Under ether anesthesia, animals of the experimental group once were installed intratracheally in the respiratory tract using a syringe 1.0 ml of the sterile saline solution containing a suspension (50 mg) of chrysotile dust - asbestos. Then, the animals were killed, their bronchial washes, centrifuged, smears from the sediment, were subsequently visualized with a microscope. Fat metabolism was assessed by the content of phospholipids in the cell, according to G.A. Merkulov. Determination of hydroxyproline in the pulmonary homogenate. The statistical differences between the two groups were assessed with the Student’s t-test. Data were expressed as mean ± SE. Probability values of p &lt;0.05 were considered significant. Results. The chronic exposure to chrysotile asbestos dust with a period of 4 months was found to causes a decrease in the activity of phagocytic cells and an increase in the destructive forms of alveolar macrophages in bronchoalveolar washes, excessive accumulation of phospholipids and an increase in oxyproline. Pneumofibrosis develops due to the cytotoxic and membrane-damaging effect of chrysotile asbestos dust. Conclusion. Thus, chrysotile asbestos dust from the Zhitikarinsky site, attributed to nanoparticles and multicomponent in chemical composition, has a cytotoxic effect, accompanied by activation of phagocytic pulmonary membrane and membrane-destructive changes in cells with accumulation of phospholipids.

Screening of additives to reduce grain damage risk on unhairing by proteinase K

Enzymatic unhairing is a cleaner strategy for leather-making. It is a potential alternative to the traditional hair-burning process. However, several shortcomings, such as uncontrolled enzymatic reaction, and risk of grain looseness and damage have restricted the broad application of enzymatic unhairing. In this work, metal ions and organic additives were screened for lessening the hydrolytic activity of proteinase K to collagen fiber. Then, the selected additives were applied to the enzymatic unhairing process for bovine hide. The results showed that a suitable concentration of metal ions (Cu (II), Fe (III) and Al (III)) and organic additives (salicylate, laurate, adipate, gallate and epicatechin (ECG)) could diminish approximately 35% of the hydrolytic activity of proteinase K to collagen fibers. Then, the additives were applied for the bovine hide enzymatic unhairing process. Hydroxyproline determination in the unhairing float shows that applying additives could reduce collagen hydrolysis. The morphology results showed that the grain damage could be significantly reduced with the addition of the screened additives in the proteinase K enzymatic unhairing system, whereas the addition of ECG and gallate significantly slowed down the unhairing speed. This outcome provides new potential to reduce the risk of grain damage in enzymatic unhairing process.Graphical abstract

Detection of Type I and III collagen in porcine acellular matrix using HPLC-MS.

Acellular matrix (ACM) has been widely used as a biomaterial. As the main component of ACM, collagen type and content show influence on the material properties. In this research, the collagen in ACM from different tissues of pig were determined by detection of marker peptides. The marker peptides of Type I and III collagen were identified from the digested collagen standards using ions trap mass spectrometry (LCQ). The relationship between the abundance of marker peptide and collagen concentration was established using triple quadrupole mass spectrometer (TSQ). The contents of Type I and III collagen in ACM from different tissues were determined. The method was further verified by hydroxyproline determination. The results showed that, the sum of Type I and III collagen contents in the ACM from small intestinal submucosa, dermis and Achilles tendon of pig were about 87.59, 81.41 and 61.13%, respectively, which were close to the total collagen contents in these tissues. The results proved that this method could quantitatively detect the collagen with different types in the ACM of various tissues.

Hydroxyproline determination for initial detection of halal-critical food ingredients (gelatin and collagen)

Gelatin and collagen are considered halal-critical ingredients as they are typically derived from either bovine or porcine animals. Current analytical methods for determining the sources of gelatin and collagen suffer from limitations in terms of robustness and false positives in peptide matching. Thus, the aim of this study was to investigate the utility of monitoring hydroxyproline, a signature amino acid for gelatin and collagen, for identifying potentially haram foodstuffs. To determine the hydroxyproline profiles among animal- and plant-based samples, one-way univariate analysis of variance followed by pair-wise comparison was used to establish statistical significance. Multivariate chemometric analysis through principal component analysis revealed a discrete distribution pattern among 59 samples due to hydroxyproline variability. Finally, inter- and intra-laboratory comparisons demonstrated the validity and robustness of hydroxyproline determination according to ISO 17025. Thus, this preliminary identification technique will aid the identification of potentially haram foodstuffs.

Quantification of Lung Fibrosis in IPF-Like Mouse Model and Pharmacological Response to Treatment by Micro-Computed Tomography.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive degenerative lung disease leading to respiratory failure and death. Although anti-fibrotic drugs are now available for treating IPF, their clinical efficacy is limited and lung transplantation remains the only modality to prolong survival of IPF patients. Despite its limitations, the bleomycin (BLM) animal model remains the best characterized experimental tool for studying disease pathogenesis and assessing efficacy of novel potential drugs. In the present study, the effects of oropharyngeal (OA) and intratracheal (IT) administration of BLM were compared in C57BL/6 mice. The development of lung fibrosis was followed in vivo for 28 days after BLM administration by micro-computed tomography and ex vivo by histological analyses (bronchoalveolar lavage, histology in the left lung to stage fibrosis severity and hydroxyproline determination in the right lung). In a separate study, the antifibrotic effect of Nintedanib was investigated after oral administration (60 mg/kg for two weeks) in the OA BLM model. Lung fibrosis severity and duration after BLM OA and IT administration was comparable. However, a more homogeneous distribution of fibrotic lesions among lung lobes was apparent after OA administration. Quantification of fibrosis by micro-CT based on % of poorly aerated tissue revealed that this readout correlated significantly with the standard histological methods in the OA model. These findings were further confirmed in a second study in the OA model, evaluating Nintedanib anti-fibrotic effects. Indeed, compared to the BLM group, Nintedanib inhibited significantly the increase in % of poorly aerated areas (26%) and reduced ex vivo histological lesions and hydroxyproline levels by 49 and 41%, respectively. This study indicated that micro-computed tomography is a valuable in vivo technology for lung fibrosis quantification, which will be very helpful in the future to better evaluate new anti-fibrotic drug candidates.

PENGARUH PEMBERIAN SALEP EKSTRAK ETANOL DAUN PILADANG(Solenostemonscutellarioides(L). Codd) SELAMA 15 HARI SECARA TOPIKAL TERHADAP AKTIVITAS PENYEMBUHAN LUKA EKSISI PADA TIKUS PUTIH JANTAN

ABSTRACTA study has been conducted on the effect of ethanolic extract of piladang leaf (Solenostemon scutellarioides (L.) Codd) topically for 15 days on excision wound healing in male white rats. This study consisted of 5 groups of each group consisting of 3 rats, ie group I as control (Vaselin Flavum), Group II as comparator (Betadine® ointment), group III, IV, and V were the group given test preparation topical with an ointment concentration of 2%, 4% and 8% extract. The experimental animal was injured, then the initial wound was measured after it was applied to the back of the mouse 2 times a day given at 8 am and 4 pm for 15 days. On the 15th day, parameters such as percentage of wound healing and hydroxyproline measurement were performed, while epithelial time was performed every day until epithelial cell was formed. The results of data analysis using one way varian (ANOVA) showed that ethanol extract of pyuridic leaf can heal wound continued duncan test (SPSS 16.0) showed that there were significant differences between groups on wound healing parameters, wound healing percentage and hydroxyproline determination (p &lt;0, 05), so it can be concluded that the preparation of leaf pyramid extract with 8% concentration more effective to give effect in wound healing.

Synthesis and biological activity of thieno[3,2-d]pyrimidines as potent JAK3 inhibitors for the treatment of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a serious and fatal lung disease, with a median survival of only 3–5 years from diagnosis. Janus kinase 3 (JAK3) has a well-established role in the pathogenesis of various autoimmune diseases, including rheumatoid arthritis (RA) and autoimmune-related pulmonary fibrosis. In this study, through the use of a conformationally-constrained design strategy, a series of thieno[3,2-d]pyrimidines were synthesized as potent JAK3 inhibitors for the treatment of IPF. Among them, the most potent JAK3 inhibitor, namely 8e (IC50 = 1.38 nM), significantly reduced the degree of airsacculitis and fibrosis according to hematoxylin-eosin (HE) staining assay for the lung tissue in the bleomycin (BLM)-induced pulmonary fibrosis mouse model. The clear reduction of the lung collagen deposition by the determination of Masson and hydroxyproline (HYP) content also demonstrated its efficacy in the treatment of fibrosis. In addition, 8e also reduced the expression of the inflammatory markers IL-6, IL-17A, TNF-α and malondialdehyde (MDA) in lung tissue, which indicated its higher anti-inflammatory activity compared with that of the reference agents (nintedanib and gefitinib). Furthermore, it possessed low cytotoxicity against normal human bronchial epithelia (HBE) cells (IC50 > 39.0 μM) and C57BL mice. All these evaluated biological properties suggest that 8e may be a potential JAK3 inhibitor for the treatment of IPF.

Topical hydrogel containing Fumaria vaillantii Loisel. extract enhances wound healing in rats

BackgroundFumaria species (Fumariacea) has traditionally been used in wound healing in Iranian folk medicine. However, with the discovery of newer agents, its use has faded off into total obscurity. This study explored the wound healing potential of a gel containing 10% Fumaria vaillantii Loisel through topical application of total extract in a model of excisional as well as incisional wound healing in albino Wistar rats.MethodsRats were anesthetized, and excisional skin wound was established using a sterilized surgical scissors. The animals were then treated with 10% F.vaillantii topical gel formulation along with the gel base. The treatments were administered once a day after the injury for 21 days. For topical treatment, the hydrogel was formulated and evaluated for chemical and physical characteristics. Histopathological analysis with hematoxylin and eosin (H&E) was used for microscopic examination of the skin tissues on 21-day-old sections of excision wound. To verify collagen formation, hydroxyproline determination was performed 21 days post wound healing. Breaking strength was determined in a 10-day-old incision wound by the uniaxial tensile test.ResultsTopical administration of F.vaillantii gel formulation significantly enhanced skin wound closure on the 6th post-wounding day compared to both gel base and the negative control, indicating an accelerated wound healing process, while a significant difference was observed on 10th and 14th post –wound days in F.vaillantii treatment compared to the negative control groups. Gel formulation prepared with a 10% F. vaillantii extract exhibited a response in terms of wound epithelialization, angiogenesis and number of hair follicles at wound area better than the gel base on the 21st post-wound day. Application of gel base produced further advantages by increasing hydroxyproline content and collagen fiber thickness. Our results on incision wound model were supported by histopathological data indicating the role of gel base in the enhancement of breaking strength.ConclusionTraditional use of Fumaria species in the skin diseases was justified in this study by revealing the increase in wound healing activity after hydrogel containing F. vaillantii total extract administration.Graphical abstract

Deletion of organic cation transporter Oct3 promotes hepatic fibrosis via upregulation of TGFβ.

Organic cation transporters (OCT) are responsible for the intracellular uptake and detoxification of a broad spectrum of endogenous and exogenous substrates. OCTs are downregulated in cholestasis, fibrosis, and hepatocellular carcinoma, but the underlying molecular mechanisms and downstream effects of OCT deletion are unknown. Oct3-knockout (Oct3-/-; FVB.Slc22a3tm10pb) and wild-type (WT; FVB) mice were subject to escalating doses of carbon tetrachloride (CCl4) or thioacetamide (TAA) for 6 wk to induce advanced parenchymal liver fibrosis. Secondary biliary fibrosis was generated by bile duct ligation. Liver fibrosis was assessed by hydroxyproline determination, quantitative Sirius red morphometry, and quantitative real-time PCR for fibrosis and inflammation-related genes. Ductular reaction was assessed by bile duct count per field of view in hematoxylin and eosin staining. General gene expression analyses were performed in liver tissue from untreated Oct3-/- and WT mice. Finally, primary murine hepatocytes were treated with the nonselective OCT inhibitor quinine, and transforming growth factor-β1 (Tgfβ1) protein expression was quantified by quantitative real-time PCR and Western blot. Oct3-/- mice developed significantly more fibrosis after bile duct ligation and CCl4 treatment compared with WT mice. Ductular reaction was enhanced in the long-term model. Concomitantly, Oct1 mRNA expression was downregulated during cholestatic and chemically (TAA and CCl4) induced fibrogenesis. The downregulation of Oct1 mRNA in fibrotic liver tissue reversed within 4 wk after TAA cessation. Gene expression analysis by next-generation sequencing revealed an enrichment of Tgfβ1 target genes in Oct3-/- mice. Tgfβ1 mRNA expression was significantly upregulated after chemically induced fibrosis (P < 0.001) in Oct3-/- compared with WT mice. Accordingly, in primary murine hepatocytes functional inhibition of OCT led to an upregulation of Tgfβ1 mRNA expression. Loss of Oct3 promotes fibrogenesis by affecting Tgfβ-mediated homeostasis in mice with chronic biliary and parenchymal liver damage and fibrosis.NEW & NOTEWORTHY We show for the first time that organic cation transporter 3 (Oct3) is not only downregulated in fibrosis but loss of Oct3 also leads to an upregulation of transforming growth factor-β contributing to fibrosis progression.

Effect of n-3 long-chain polyunsaturated fatty acids on wound healing using animal models – a review

The present review summarizes results of experiments, mostly performed on rodents, regarding the effects of fish oil (FO) and its biologically active constituents, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on the healing of cutaneous wounds, but also of selected other types of injury. Structure, metabolism and functions of EPA/DHA in an organism are briefly mentioned, with an emphasis on the ability of these long-chain polyunsaturated fatty acids to modulate inflammation. Wound healing as a complex programmed sequence of cellular and molecular processes including inflammation, cell migration, angiogenesis, synthesis of provisional matrix, collagen deposition and reepithelialisation is briefly described. Markers for evaluation of the healing process include planimetry indices, tensile strength, quantification of collagen synthesis including hydroxyproline determination, histopathology/immunohistochemistry and genomic/proteomic markers. As far as effects on wound healing are concerned, the main emphasis is put on the outcomes of experiments using a dietary FO/DHA/EPA administration, but the results of experiments with a parenteral application are also mentioned, together with selected relevantin vitrostudies. An important conclusion from the above-mentioned studies is an inconsistency of FO/DHA/EPA effects on wound healing: decreased/increased collagen deposition; lower/higher counts of the inflammatory cells in the healing tissue; increased/decreased concentration of both pro- and anti-inflammatory cytokines; DHA accelerated/delayed wound healing process. Some experiments indicate superiority of DHA over EPA regarding wound healing.

Effect of Proanthocyanidin-enriched extracts on the inhibition of wear and degradation of dentin demineralized organic matrix

ObjectivesThe aim of this study was to evaluate the effect of Cranberry and Grape seed-enriched extract gels in inhibiting wear and degradation of demineralized organic matrix (DOM). Design225 dentin specimens obtained from bovine incisors were randomly allocated into 5 groups (n=45): 10% Grape seed extract gel (GSE), 10% Cranberry extract gel (CE), 0.012% Chlorhexidine gel (CX), 1.23% NaF gel (F), and no active compound gel (P, placebo). Before the treatments, samples were demineralized by immersion in 0.87M citric acid, pH 2.3 (36h). Then, the studied gels were applied once over dentin for 1min. Next, the samples were immersed in artificial saliva containing collagenase obtained from Clostridium histolyticum for 5days. The response variable for dentin wear was depth of dentin loss measured by profilometry and for collagen degradation was hydroxyproline determination. Data were analyzed by ANOVA followed by Tukey's test and Pearson Correlation Test (p<0.05). ResultsGrape seed extract significantly reduced dentin wear compared to the other groups (p<0.05). Cranberry extract and Chlorhexidine did not differ statistically and were able to reduce wear when compared to NaF and placebo treatments. The hydroxyproline analysis showed that there was no significant difference among groups for all treatments (p<0.05). Correlation analysis showed a significant correlation between the amount of degraded DOM evaluated by profilometry and the determination of hydroxyproline. ConclusionCranberry extract was able to reduce the dentin wear and collagen degradation, likely due to the proanthocyanidin content and its action. Therefore, Cranberry could be suggested as an interesting natural-based agent to prevent dentin erosion.

Effects of connective tissue growth factor on human periodontal ligament fibroblasts

ObjectiveThe aim of this study was to evaluate the effects of different concentrations of connective tissue growth factor (CTGF) on human periodontal ligament fibroblasts(HPLFs). DesignHPLFs were cultured and identified. Then, different concentrations of CTGF (1, 5, 10, 50, 100ng/ml) were added to the HPLF culture. Next, CCK-8 assays, alkaline phosphatase (ALP) assays, hydroxyproline determination, alizarin red staining methods, Transwell chambers and real-time PCR methods were applied to observe the effects of CTGF on the proliferation, ALP activity, synthesis of collagen, formation of mineralized nodules and migration. We also studied expression of ALP, fiber link protein (FN), integrin-binding sialoprotein (IBSP), osteocalcin (OC), and integrin beta 1 (ITGB1) mRNA by HPLFs. Statistical significance was assumed if P<0.05 or P<0.01. ResultsThe addition of CTGF (1, 5, 10ng/ml) remarkably promoted the proliferation and collagen synthesis of HPLFs compared with controls. CTGF (1, 5, 10, 50ng/ml) improved ALP activity of HPLFs, and at all concentrations, CTGF (1, 5, 10, 50, 100ng/ml) improved the expression of ALP, FN, IBSP and ITGB1 mRNA. In addition, CTGF (1, 5, 10, 50, 100ng/ml) promoted the migration of HPLFs, which was dose-dependent, with maximal promotion in the 10ng/ml group (P<0.05 or P<0.01). ConclusionsThus, in a certain range of concentrations, CTGF can promote the biological effects, including proliferation, migration and collagen synthesis of HPLFs, to promote the differentiation of HPLFs in the process of osteogenesis.

Bioanalytical Method for the Determination of Hydroxyproline in Mouse Kidney by High-Performance Liquid Chromatography with Tandem Mass Spectrometric Detection.

An analytical method was developed and validated for the measurement of hydroxyproline (Hyp) levels in mouse kidney by high-performance liquid chromatography with tandem mass spectrometric detection (LC/MS/MS) using an analytical column specially designed for the LC/MS/MS analysis for intact amino acids. Tissue hydrolyzed with hydrochloric acid could be directly injected into the LC/MS/MS, as well as separated and detected using the deuterium labelled Hyp as an internal standard. The calibration curve showed good linearity from 5 to 500 nmol/mg of tissue; the precision and accuracy, including within- and between-run, were less than 6% and within 100 ± 6%, respectively.

Protective Effects of Hydrogen-Rich Saline Against Lipopolysaccharide-Induced Alveolar Epithelial-to-Mesenchymal Transition and Pulmonary Fibrosis.

BackgroundFibrotic change is one of the important reasons for the poor prognosis of patients with acute respiratory distress syndrome (ARDS). The present study investigated the effects of hydrogen-rich saline, a selective hydroxyl radical scavenger, on lipopolysaccharide (LPS)-induced pulmonary fibrosis.Material/MethodsMale ICR mice were divided randomly into 5 groups: Control, LPS-treated plus vehicle treatment, and LPS-treated plus hydrogen-rich saline (2.5, 5, or 10 ml/kg) treatment. Twenty-eight days later, fibrosis was assessed by determination of collagen deposition, hydroxyproline, and type I collagen levels. Development of epithelial-to-mesenchymal transition (EMT) was identified by examining protein expressions of E-cadherin and α-smooth muscle actin (α-SMA). Transforming growth factor (TGF)-β1 content, total antioxidant capacity (T-AOC), malondialdehyde (MDA) content, catalase (CAT), and superoxide dismutase (SOD) activity were determined.ResultsMice exhibited increases in collagen deposition, hydroxyproline, type I collagen contents, and TGF-β1 production in lung tissues after LPS treatment. LPS-induced lung fibrosis was associated with increased expression of α-SMA, as well as decreased expression of E-cadherin. In addition, LPS treatment increased MDA levels but decreased T-AOC, CAT, and SOD activities in lung tissues, indicating that LPS induced pulmonary oxidative stress. Hydrogen-rich saline treatment at doses of 2.5, 5, or 10 ml/kg significantly attenuated LPS-induced pulmonary fibrosis. LPS-induced loss of E-cadherin in lung tissues was largely reversed, whereas the acquisition of α-SMA was dramatically decreased by hydrogen-rich saline treatment. In addition, hydrogen-rich saline treatment significantly attenuated LPS-induced oxidative stress.ConclusionsHydrogen-rich saline may protect against LPS-induced EMT and pulmonary fibrosis through suppressing oxidative stress.

Determination of hydroxyproline in liver tissue by hydrophilic interaction chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry

A method for the determination of hydroxyproline (Hyp) in liver tissue of mice by hydrophilic interaction chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry (HILIC-HRMS) was developed. The liver tissue samples of normal mice and liver fibrosis mice induced by carbon tetrachloride were hydrolyzed by concentrated hydrochloric acid. After filtrated and diluted by solution, the diluent was separated on an Hypersil GOLD HILIC column (100 mm×2.1 mm, 3 μm). Water-acetonitrile (28:72, v/v)were used as the mobile phases with isocratic elution. Finally, the target analytes were detected in positive model by HRMS equipped with an electrospray ionization source. The linear range of hydroxyproline was from 0.78 to 100.00 μg/L with the correlation coefficient (R2) of 0.9983. The limit of quantification was 0.78 μg/L. By detecting the spiked samples, the recoveries were in the range of 97.4%-100.9% with the relative standard deviations (RSDs) between 1.4% and 2.0%. In addition, comparison of the measurement results by this method and the chloramine T method was proceeded. It was found that the linear correlation between the two methods was very good, and the Pearson correlation coefficient was 0.927. And this method had simpler operation procedure and higher accuracy than chloramine T method. This method can be used for the quick determination of hydroxyproline in liver tissue samples.